calpain and Anemia--Sickle-Cell

Topics: Anemia, Sickle Cell; Calcium; Calmodulin; Calpain; Cytoskeletal Proteins; Erythrocyte Membrane; Erythrocytes; Erythrocytes, Abnormal; Gene Expression Regulation; Humans; Malaria; Membrane Lipids; Membrane Proteins; Phosphatidylinositols; Protein Kinases; Protein Processing, Post-Translational; Receptors, Cell Surface; Signal Transduction; Sulfhydryl Compounds

Topics: Anemia, Sickle Cell; Blood Platelets; Calpain; Humans; Platelet Activation; Thrombosis

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5'-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, and partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa (α

Topics: Anemia, Sickle Cell; Animals; Blood Coagulation; Blood Platelets; Calpain; Disease Models, Animal; Female; Humans; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Activation

Renal medullary carcinoma (RMC) is a rare and highly aggressive neoplasm that most often occurs in the setting of sickle cell trait or sickle cell disease (SCD). Most patients present with metastatic disease resistant to conventional chemotherapy, and therefore there is an urgent need for molecular insight to propose new therapies.. To determine the molecular alterations and oncogenic pathways that drive RMC development.. A series of five frozen samples of patients with RMC was investigated by means of gene expression profiling, array comparative genomic hybridization, and RNA and whole exome sequencing (WES).. RNA and DNA sequencing read data were analyzed to detect gene fusions and somatic mutations. Gene fusions mutations were validated by real-time polymerase chain reaction and fluorescence in situ hybridization. Gene expression profiling was analyzed by unsupervised hierarchical clustering and Gene Set Enrichment Analysis (Broad Institute, Cambridge, MA, USA).. We observed inactivation of the tumor suppressor gene SMARCB1 in all tumors. In all four cases developed in patients with SCD, we identified an original mechanism of interchromosomal balanced translocations that disrupt the SMARCB1 sequence and thus contribute to its inactivation. Gene expression profiling revealed that RMC shares common oncogenic pathways with pediatric malignant rhabdoid tumors, another tumor subtype characterized by SMARCB1 deficiency.. RMCs are characterized by an original mechanism of interchromosomal balanced translocations that disrupt the SMARCB1 sequence. WES reveals that RMCs show no other recurrent genetic alteration and an overall stable genome, underscoring the oncogenic potency of SMARCB1 inactivation.. Our comprehensive molecular study supports a pivotal role of the tumor suppressor gene SMARCB1 in the development of renal medullary carcinoma. The use of therapeutic strategies based on the biologic effects of its inactivation should now open new perspectives for this typically lethal malignancy.

Topics: Adolescent; Adult; Anemia, Sickle Cell; Calpain; Carcinogenesis; Carcinoma; Child; Comparative Genomic Hybridization; DNA-Binding Proteins; Exome Sequencing; Gene Expression Profiling; Gene Fusion; Humans; Kidney Neoplasms; Nuclear Proteins; Nuclear Receptor Subfamily 1, Group F, Member 1; RNA, Long Noncoding; Sequence Analysis, RNA; SMARCB1 Protein; Trans-Activators; Transcription Factors; Translocation, Genetic

Topics: Anemia, Sickle Cell; Animals; Calpain; Disease Models, Animal; Gene Deletion; Gene Silencing; Humans; Mice; Mice, Knockout; Pain

Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD-related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain-1 in favor of activator in dense sickle cells. Calpain-1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain-1, BDA-410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA-410 improved RBC morphology, reduced RBC density (D(20); from 1106 ± 0.001 to 1100 ± 0.001 g/ml; P<0.05) and increased RBC-K(+) content (from 364 ± 10 to 429 ± 12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca(2+)-activated K(+)channel (Gardos channel), and decreased membrane association of peroxiredoxin-2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA-410 treatment, which suggests that calpain-1 inhibition affects the PKC-dependent fraction of the Gardos channel. BDA-410 prevented hypoxia-induced RBC dehydration and K(+) loss in SAD mice. These data suggest a potential role of BDA-410 as a novel therapeutic agent for treatment of SCD.

Topics: Anemia, Sickle Cell; Animals; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Dehydration; Disease Models, Animal; Erythrocytes, Abnormal; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice; Mice, Mutant Strains; Mice, Transgenic; Sulfonamides

Calpain, a calcium-dependent, neutral cysteine-protease was purified from the erythrocyte cytosol of subjects having essential hypertension (HTN), sickle cell anaemia, (SCA), or kwashiorkor (KWA). Identical electrophoretic mobility on SDS-polyacrylamide gradient gel, sensitivity to micromolar amounts of Ca2+, absolute requirement for a reducing environment and a high susceptibility to inhibition by leupeptin and thiol-group modifying reagents confirm that calpain preparations from these erythrocytes are equivalent to calpain I. Whereas the extent of calpain activation of erythrocyte membrane Ca2(+)-pumping ATPase of normal subjects was almost equal to that due to calmodulin, calpain activation of the HTN and SCA pump was greater than activation by calmodulin. Like in normal membranes, exogenous calmodulin protected the Ca2(+)-pumping ATPase of these erythrocytes against calpainization; the degree of protection by calmodulin is least in SCA and HTN. Electrophoretic separation of erythrocyte membranes and the purified Ca2(+)-pumping ATPase of HTN, SCA and KWA subjects does not indicate the presence of fragments resulting from the proteolytic action of calpain.

Topics: Anemia, Sickle Cell; Calcium-Transporting ATPases; Calpain; Enzyme Activation; Erythrocyte Membrane; Erythrocytes; Humans; Hypertension; In Vitro Techniques; Kwashiorkor